KR20230062601A - Automated Method for Direct Sampling of Immune Cells from Whole Blood or Other Biological Samples in Microwell Plates - Google Patents

Abstract

A method for automatically sampling immune cells from a biological fluid sample, eg, whole blood, deposited in the wells of a microwell plate. The sample contains red blood cells (RBCs) and magnetic beads designed to bind to red blood cells. The microwell plate is placed on a shaker with a magnetic adapter comprising at least one magnet. The magnet causes the RBCs bound to the magnetic beads to be attracted to the walls of the well (eg, the bottom or sidewalls) to move and to be held against the walls. The shaker then substantially uniformly or homogenously suspends the immune cells in the biological fluid sample within the region of the well, but still maintains retention of the RBCs on the walls of the well such that the immune cells are substantially isolated from the RBCs within the region of the well. and to agitate the microwell plate for a period of time in such a manner as to During or after shaking, the sample probe is then lowered into an area of the well to withdraw a portion of the sample containing immune cells.

Description

Automated Method for Direct Sampling of Immune Cells from Whole Blood or Other Biological Samples in Microwell Plates

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to US Provisional Application Serial No. 17/009,225, filed September 1, 2020, which is incorporated herein by reference.

The present invention relates to the production of immune cells (lymphocytes, neutrophils, monocytes and macrophages, or leukocytes in general) from a biological fluid sample loaded into the wells of a microwell plate. It relates to a method for directly sampling cells such as leukocytes or white blood cells (WBCs). One specific application for the method of the present invention is the direct sampling of immune cells from whole blood samples. The method is also suitable for direct sampling of immune cells from other biological fluid samples that may contain red blood cells (RBCs), such as cyst fluid samples, amniotic fluid samples, bone marrow samples or cerebrospinal fluid samples.

The term “microwell plate” refers to a flat plate form that forms an array of many small individual sample-holding wells, typically 6, 12, 24, 48, 96, 384 or more wells per plate. It is used to refer to a test device format, or a test device format in the form of a test tube array, typically 40 test tubes in an array. The term is sometimes referred to in the art as "microtiter plate" or "microplate".

Such microwell plates are typically used in conjunction with a sample handling device, which automatically extracts a portion of a sample from one of the wells and transfers the sample to an analytical instrument, e.g., one or more measurements of the extracted sample. It is introduced into a flow cytometer, hematology analyzer, cell sorter, mass spectrometer, etc.

Sampling whole blood with a cytometer for white blood cell (WBC) analysis is difficult because whole blood tends to clog small flow paths. In addition, it is known that the WBC and RBC populations are difficult to distinguish in a conventional cytometer. Therefore, the art is developing methods for removing RBCs from whole blood samples. Red blood cell lysis (RBC lysis) uses a buffer solution such as ammonium chloride that lyses red blood cells while minimizing the effect on white blood cells. The use of traditional RBC lysis methods in the microplate format used for processing whole blood samples is labor intensive, which creates RBC debris that can clog the cytometer flow cell and is very dirty and significantly increases carryover from one sample to another. In addition, the RBC lysis method may cause loss of data integrity since the hypotonic buffer used for lysis is net physiological and may affect normal immune cell activity. .

Another method, traditional gradient centrifugation, can be used for purification of WBCs, and this method is used for samples in test tube format, but not in microplate format.

Accordingly, there is a need in the art for a method of automatically sampling immune cells from samples containing RBCs or other biological fluid samples in a microwell plate format.

In one aspect of the invention, a method for automatically sampling cells, such as immune cells, from a biological fluid sample deposited in a well of a microwell plate having walls (ie, bottom walls or side walls) is provided. The sample contains, for example, (1) RBCs and (2) magnetic beads conjugated to antibodies or otherwise designed to bind RBCs in the sample. The method comprises the steps of a) placing a microwell plate on a shaker having a magnetic adapter comprising a magnet, the magnet attracting the RBC coupled to the magnetic bead to the wall of the well; moving to the wall and being held against the wall; b) using the shaker to substantially uniformly or homogeneously suspend the immune cells in the biological fluid sample in the region of the well, wherein the immune cells are isolated from the RBCs in the region of the well shaking for a period of time and in a manner so as to maintain retention of the RBCs to the wall of the well such that ); and c) lowering the sample probe into the well in the region of the well and withdrawing a portion of the sample containing the immune cells from the region.

In another aspect, a shaker system is described in the form of a shaker having a top surface configured to shake a microwell plate in a controlled and programmable manner. The agitator includes a magnetic adapter that cooperates with a structure on the agitator to removably fit the top surface of the agitator. The magnetic adapter is in the form of a substantially flat structure holding an array of individual magnets, the magnetic adapter being fitted onto the top surface of the shaker and sandwiched between the top surface of the shaker and the microwell plate. It consists of

In one configuration, an array of magnets is arranged on the magnetic adapter to register with the bottoms of the wells of the microwell plate when the microwell plate is placed on top of the magnetic adapter.

In another embodiment, an apparatus includes a control system for an agitator. The control system includes a shaker that substantially uniformly or homogenously suspends the immune cells in the biological fluid sample within the region of the well, wherein the immune cells are substantially isolated from the RBCs within the region of the well, wherein the RBCs are magnetically coupled to the walls of the well. The shaker is operated to agitate the microwell plate for a period of time in such a way as to maintain retention.

In another aspect, a robotic sampling probe, a shaker having an upper surface, and a magnetic adapter designed to cooperate with structures on the shaker to removably fit into the upper surface of the shaker. ) A flow cytometer comprising a is provided. Additionally, the magnetic adapter has one or more features to retain the microwell plate disposed thereon. The magnetic adapter may take the form of a substantially flat structure holding an array of individual magnets and is configured to be interposed between the shaker and the microwell plate. A flow cytometer includes a control system for a shaker configured to agitate a microwell plate for a period of time in a manner to substantially evenly or homogeneously suspend cells, such as immune cells, within a biological fluid sample disposed within the wells of the microwell plate. include The immune cells float within the region of the well, but still retain retention of the RBCs magnetically coupled to the wall of the well due to the one or more magnets, such that the immune cells are substantially isolated from the RBCs within the region of the well. The flow cytometer also includes analytical instrumentation for counting, sorting, or performing other measurements on immune cells drawn from the wells of the microwell plate, wherein the probes draw the immune cells from the wells. and introduce immune cells into the device.

1A-1E are illustrations of a workflow or method for automatically sampling cells, such as immune cells, in a microwell plate format, from a biological fluid sample containing red blood cells, e.g., a whole blood sample, which is illustrated in FIGS. 1A and 1E. It consists of a preliminary step shown in Fig. 1b, followed by steps shown in Figs. 1c, 1d and 1e.
FIG. 2 is a view of a microwell plate positioned over the shaker according to FIG. 1, secured to the shaker and showing a magnetic adapter positioned between the shaker and the microwell plate.
Figure 3 is an exploded view of the assembly of Figure 2;
Fig. 4 is a plan view of the magnetic adapter of Figs. 2 and 3;
Fig. 5 is a side view of the magnetic adapter of Fig. 4;
FIG. 6 is a diagram illustrating that a sampling probe inserted into one of the wells of the microwell plate of FIG. 2 takes out a sample from the well according to step E of FIG. 1E.
FIG. 7 is an exploded view of the magnetic adapter of FIG. 4, showing an optional ferromagnetic shield disposed adjacent and directly below the bottom surface of the magnetic adapter.
Figure 8 is a partial cross-sectional view of the assembly of Figure 7;
Figure 9 is an illustration of a flow cytometer configured for processing samples, including whole blood samples, in a microwell plate format comprising the shaker and magnetic assembly of Figure 2; 9 also shows the associated workstation used to display analysis results from the flow cytometer.
Figure 10 is a perspective view of the flow cytometer of Figure 9; The shaker is moved into the flow cytometer in position for the various processing steps to be performed on the microwell plate; 10 also shows a sampling probe connected to a three-axis robotic movement system that allows all wells to be sampled.
11 is another perspective view of the flow cytometer of FIG. 9 with a sample probe in position for withdrawing a sample from one of the wells according to step E of FIG. 1E.

outline

In one aspect of the present disclosure, a method for automatically sampling cells, such as immune cells, from a biological fluid sample containing red blood cells (RBCs) deposited in wells of a microwell plate is provided. In the description below, the fluid sample is described as whole blood, but as noted above, the sample may be red blood cells, other cell types, or other biological fluids containing mixed particle populations such as particles of interest, etc. . These fluids may be, for example, amniotic fluid, cerebrospinal fluid, and the like.

The methodology is shown in FIGS. 1A-1E, consisting of preliminary steps shown in FIGS. 1A and 1B, followed by steps 1C, 1D, and 1E. This process is explained in conjunction with FIGS. 2 and 3 . From the description below, it will be understood that the process shown for a single well 10 of a microwell plate 100 (FIG. 2) is performed for all wells loaded with sample 12 (eg, whole blood). will be.

In step A (FIG. 1A), a custom treatment and/or staining cocktail (reagent) is added to the sample 12. These reagents include marker beads for well-ID for samples with few white blood cells (WBCs), in-well counting beads to facilitate WBC counting, staining agent, or possibly others.

In step B ( FIG. 1B ), magnetic beads 14 conjugated to antibodies or otherwise designed to bind to RBCs 15 in the sample are added to the sample 12 . An example of such beads are anti-human CD235 antibody-coated magnetic beads. Antibodies coated on magnetic beads are not limited to antibodies against the CD235 molecule, and may also be other antibodies against molecules specifically expressed on RBCs other than WBCs. In addition, the moiety molecule coated on the beads is not limited to antibodies, but antibody fragments, affimers, and antigens that can specifically bind to molecules expressed only on RBCs and not on WBCs. may be other molecules such as

Steps A and B can be performed on a laboratory bench prior to inserting the microwell plate 100 (FIG. 2) into the assay instrument. After step B, the microwell plate 100 may be incubated for a period of time, for example 1 minute to 2 hours, to allow the magnetic beads 14 to be conjugated to the RBCs 15; The duration for incubation is flexible and can be optimized by the user.

In step C ( FIG. 1C ), magnetic separation of RBCs 15 from WBCs 17 in sample 12 is performed. In particular, the microwell plate 100 is placed on top of a microtiter plate shaker 104, such as a BioShake 3000, and the shaker 104 has a magnet ( 120). The magnet 120 attracts the magnetic beads 14, and the RBCs 15 coupled to the magnetic beads 14 are attracted to the bottom wall 16 or side wall 19 of the well 10 and move, and the wall ( 16) (or the wall 19 depending on the position of the magnet) to hold it. This action separates the WBCs 17 into a layer above the RBCs 15 bonded to the bottom wall 16 of the well 10. The magnetic adapter 102 is placed on top of the microtiter plate shaker and is designed to be removed from or inserted onto the top surface of the shaker 104 by the user without the use of special tools. Thus, the adapter 102 is interposed between the top of the shaker 104 and the microwell plate 100. No shaking is performed in step C, or optionally only very moderate shaking can be performed in step C, which, depending on the position of the magnets, causes the magnetic beads 14 to bind to the RBCs 15 and , continuing to allow the RBCs 15 to be pulled down the sidewall 19 and/or bottom wall 16 of the well 10. In embodiments where the magnets are positioned below the microwell plate, the RBCs are pulled down to the bottom wall 16 as shown in FIG. 1C.

In step D (FIG. 1D), after the RBCs 15 are seated in the well bottom 16 as shown in FIG. 1D, the shaker 104 is turned on and the upper liquid layer (mostly serum and plasma) ) substantially uniformly or homogeneously resuspends the WBCs (17), while the RBCs (15) are firmly attached to the bottom of the well (16) by the attraction of the conjugated magnetic beads (14) to the magnet. Shake at a specified speed (revolutions per minute, rpm) within a specified range so that the That is, at this stage the shaker 104 substantially uniformly or homogenously suspends and distributes the immune cells 17 (WBCs) in the biological fluid sample within the area of the well 18, but not the immune cells 17. ) (WBC) constant in a manner to maintain retention of the RBCs relative to the bottom wall 16 and/or sidewall 19 (FIG. 1C) of the well 10 such that the WBCs are substantially isolated from the RBCs in the region 18. Shake the microwell plate 100 for a period of time. The manner in which agitation is performed (i.e., speed) is described in detail below, the details of which include the strength of the beads 14 binding to the RBCs 15, the design of the agitator 104 itself, the microwell plate 100 This can vary depending on the design, liquid volume in each well, and can be determined empirically for a particular magnetic bead and given shaker. After some predetermined period of time, for example 10 seconds, the agitator 104 is turned off.

In step E ( FIG. 1E ), software having a specific sampling protocol governing the operation of the sampling probe 200 immediately after shaking ends, or optionally while shaking is in progress, causes the sample probe 200 to move its tip upwards. It is automatically guided to insert into the liquid layer (region 18) and acquire a specific volume of WBCs 17 without contamination of the RBCs 15 that are still magnetically held at the bottom of the well 16. This acquisition is performed by controlling the sampling height position of the sample probe 200 . Accordingly, in this step, the sample probe 200 is lowered into the well 10 within the region 18 of the well in which the WBC 17 is suspended substantially uniformly or homogeneously. A portion of the sample containing immune cells is then withdrawn from region 18 . This is shown in FIG. 6 .

After withdrawal of the WBC sample 17, the sample may be processed in an analytical instrument to which the probe 200 belongs, such as a flow cytometer. A sample is introduced into an analytical device, such as a sample introduction port, which delivers the sample to a further analytical device that performs measurements on the sample.

Shaker and Magnetic Adapter Design

Referring now to Figures 2-5, shaker 104 is any commercially available device designed to shake or agitate microwell plates, such as the aforementioned shaker, from companies such as Qlnstruments, ThermoFisher, SigmaAldrich, Southwest Science, and the like. can take the form of an agitator from Shaker 104 is typically designed with eccentric or orbital rotation to induce vortex action within the well, and is programmatically controlled to rotate at a predetermined or user-specified speed. For example, the eccentric offset can be 2 mm, and a desired uniform distribution of WBCs 17 is achieved without ejection of RBCs 15 from the bottom of the wells, for example at 250 rpm for 10 seconds in the wells. The optimum rotational speed and duration to be achieved in the upper region 18 is determined.

Referring to FIG. 3 , the magnetic adapter 102 may take the form of a substantially flat structure as shown holding individual magnets 120 , one for each well 10 of the microwell plate 100 . . Alternatively, magnets 120 on adapter plate 102 may be configured as two or more magnets per well, or even two or more wells per magnet or group of magnets. A configuration with exactly one magnet per well is not required, just that the magnets produce a substantially uniform magnetic field. The magnetic adapter 102 is configured to fit onto the upper surface 106 of the shaker 104 and is configured to be sandwiched between the upper surface 106 and the microwell plate 100 as shown in FIG. 2, A magnet 120 holds the RBC 15 to the bottom wall of the well. The adapter plate 102 includes corner features 114 designed to retain the corners 116 of the microwell plate 100 and prevent the plate from dislocating from the shaker 104 during agitation. include Additionally, corner features 114 may be designed to guide microwell plate 100 into the correct position when a user places the microwell plate onto adapter 102 . The adapter 102 is designed to be easily attached to and removed from the shaker 104 manually without the use of tools. This is done, for example, by resilient gripping skirts 110 that descend from adapters 102 that fit against recesses 112 provided on the front and rear sides of agitator 104. can be achieved A locating pin 108 protrudes from the upper surface of the agitator 104, which fits into a corresponding recess provided in the adapter plate 102 and snugly fits the adapter plate as shown in FIG. (snugly) It serves to grip. Depending on the embodiment, the locating pins 108 may grip and hold the adapter plate 102 in place, or the microwell plate 100, or both.

The locating pins 108 may be computer controlled to move into and out of a gripping position to grip the adapter plate 102 . This feature can be important when the shaker 104 is used in an automated mode. In this mode, the software automatically positions the locating pins 108 when the robotic arm removes the microwell plate 100 from the shaker 104 and/or places a new microwell plate on the shaker. can be loosened or tightened with When the magnetic adapter design includes a magnetic shield 302 (see description of FIGS. 7 and 8 ), the existing gripper/locating pins 108 on some shakers (e.g. BioShake (3000)) is not long enough to reach the microwell plate and hold it in place. One solution to this is to add additional compliant gripping features (not shown) to the magnetic adapter. These compliant gripping features interact with the existing gripper/locating pins 108 on the BioShake 3000 so that when the locating pins 108 of the BioShake 3000 are closed, the compliant gripping features grip the microwell plate. push to do it. Then, when the BioShake 3000 positioning pins 108 are relaxed, the compliant gripping features on the magnetic adapter are also relaxed and the microwell plate is released. Of course, these solutions can be applied to other agitator designs; Indeed, the specific form factor and design of the magnetic adapter 102 of FIG. 3 may be changed to be compatible with the specific configuration of the microwell plate 100 as well as the surface geometry, locating pins and grippers of the shaker in question. It can be.

One possible configuration of adapter plate 102 and magnets 120 is shown in the exploded view of FIG. 7 . Adapter plate 102 has an array of recessed pockets 300, each receiving and holding one of magnets 120. The layout of the array of pockets 300 (and thus magnets 120) is designed such that each magnet is positioned directly beneath a sample well in a standard format microwell plate. Although Figure 7 shows a 96 well plate, the same design considerations would apply to a microwell plate with a different number of wells, such as a 384 well plate. Alternatively, magnets 120 on adapter plate 102 may be configured as two or more magnets per well, or even two or more wells per magnet or group of magnets. A configuration with exactly one magnet per well is not required, just that the magnets produce a substantially uniform magnetic field.

Shaker 104 is typically configured with a home position sensor, which may take the form of, for example, a hall-effect sensor. The presence of magnets on the adapter plate 102 directly above the shaker 104 creates a magnetic field that can interfere with the operation of the home position sensor within the shaker. To ameliorate this, in one optional embodiment, a ferromagnetic shield 302 ( FIGS. 7 and 8 ) in the form of a thin, flat plate of ferromagnetic material such as stainless steel or mu-metal is provided. Positioned below the adapter plate 102 as shown in FIGS. 7 and 8 , when the adapter plate 102 and shield 302 are placed on the shaker 104, the home position sensor operation is not adversely affected. do not receive Shield 302 may be mounted or attached to the bottom surface of magnetic adapter plate 102 in any suitable manner. Shield 302 redirects the magnetic field lines and reduces the field strength at the location of the home position sensor. The shield thickness and material are not particularly critical if made of a ferromagnetic material. However, increasing the separation distance between the agitator 104 and the magnets 120 and adding a shield 302 under the magnets 120 allows the agitator to operate correctly.

example

Flow cytometer for direct sampling of immune cells from whole blood samples in microwell plates (FIG. 1, 9-11)

Examples of systems in which the method may be performed are shown in FIGS. 9-11 . FIG. 9 is an illustration of a flow cytometer 400 configured for processing a sample comprising a whole blood sample in a microwell plate form 100 comprising the shaker 104 and magnetic adapter 102 assembly of FIG. 2 . The instrument shown is the assignee's iQue® 3 flow cytometer, but the principles discussed in this example will apply to other assay devices or flow cytometers. 9 also shows an associated workstation 410 used to display analysis results from the flow cytometer. Workstation 410 allows the user to configure specific workflows and processing steps to be performed for a given microwell plate, and associated samples loaded into microwell plate 100, such as launching the "whole blood sample module" described below. It may contain interfaces that allow it to be specified. The flow cytometer may also include a supply 402 of reagents, buffers, cleaning solutions, and the like. The flow cytometer 400 includes a loading station 404 where the microwell plate 100 is placed on the shaker 104, a transparent plastic partition 412 mounted on a hinge that opens to allow access to the interior of the flow cytometer. , and rinse station 406 (FIG. 10).

FIG. 10 is a perspective view of the flow cytometer 400 of FIG. 9 , showing the transparent partition 412 moved to an open position and placed in a rinse station 406 where various processing steps may be performed on the microwell plate 100. Shaker 104 is shown moved into an adjacent flow cytometer 400. 10 also shows a sampling probe 200 coupled to a three-axis robotic movement system 414 . The movement system 414 is under program control to move in the X, Y and Z directions, thus allowing all wells on the microwell plate 100 to be sampled by the sampling probes 200.

The flow cytometer 400 also includes an assay instrument for performing flow cytometry on immune cells taken from the wells of the microwell plate 100 located behind the panel of the flow cytometer shown in FIG. 10, which is conventional; Therefore, for the sake of brevity, it is not described in detail in this document. The probe 200 extracts immune cells from the well and introduces the immune cells into an assay instrument.

11 is another perspective view of the flow cytometer of FIG. 9 with a sample probe 200 in position for withdrawing a sample from one of the wells in the microwell plate 100 according to step E of FIGS. 6 and 1E.

The flow cytometer 400 includes a loading station 404 (FIG. 9), which allows the user to load the microwell plate 100 onto the shaker 104 (shaker 104 as shown in FIG. 2). with the magnetic adapter 102 located on the upper surface of the Shaker 104 is connected to a motor-driven electro-mechanical system, which in turn moves shaker 104 from loading station 404 (FIG. 9) through an opening in transparent partition 412 into the interior of the flow cytometer. (see FIG. 10).

According to the method of FIG. 1 , steps A and B of FIGS. 1A and 1B , respectively, are typically performed off-line, for example on a laboratory bench. After step B, the microwell plate 100 is then placed on the shaker 104 at the loading station 404 (see FIG. 9). With the magnetic adapter 102 interposed between the shaker 104 and the microwell plate 100, when the microwell plate is placed on the magnetic adapter, according to step C of FIG. is pulled down to the bottom of During or immediately after performing step C, which may be, for example, 30, 60 or 90 seconds in duration, the shaker is moved from the loading station 404 of FIG. 9 to the position shown in FIGS. 10 and 11 of the flow cytometer. are moved inside

In step D of FIG. 1D, the shaker 104 is activated for a period of time while the RBCs are suspended to substantially uniformly or homogeneously suspend the immune cells in the sample at the top of the wells in area 18 (FIG. 1D). It will be held against the floor and/or side wall. This shaken step may be performed with the shaker 104 at the loading station 404 of FIG. 9 or at the sample acquisition location shown in FIG. 10 .

In step E of FIG. 1E , the shaker 104 is stopped, and immediately thereafter, the sample probe 200 is lowered into the first well of the microwell plate as shown in FIG. 11 , substantially according to FIG. 6 . A sample containing only immune cells is withdrawn. The sample probes are actuated by the robotic movement system in the X, Y and Z directions to sample in the same manner as any and/or all wells of the microwell plate 100 that hold the samples. After the sample is acquired by the probe 200 using force from a peristaltic pump or a vacuum pump (not shown), the sample is transferred to an analysis instrument (not shown) within the flow cytometer 400; It performs flow cytometry in a conventional manner. For example, the flow cytometer 400 of FIG. 9 is described in U.S. Patents 10,048,191; 9,897,531; 9,797,917; and 6,890,487.

software behavior

The operation of the shaking and sampling mode of FIGS. 1D and 1E in the flow cytometer of FIGS. 9-11 controls the operation of the shaker 104, the sample probe 200, and the associated three-axis robotic movement system 414 ( 400) is controlled by processors and software operations within it. This operation is within a mode or module of the flow cytometer designed to process whole blood samples, and is referred to as the “whole blood sample module” or “whole blood sample mode” in the following discussion. The parameters of this mode may be accessed by the workstation 410 of FIG. 9 and configured by the user, or may be preset commands that are automatically executed when the user selects a whole blood sample module for processing of a given microwell plate. can

Module detection and RBC pull down

In FIG. 10 , prior to sampling, the probe ( FIGS. 10 , 200 ) extending from the sampling head 411 ( FIG. 10 ) is lowered until it contacts the magnetic adapter 102 . When the probe 200 senses a difference in deck height (due to the presence of the magnetic adapter 102 on top of the shaker), the software within the flow cytometer is triggered to alert the user that the system is operating in the whole blood sample module or mode. . A predefined waiting period for RBC pulldown is automatically included in this mode, step C of Fig. 1c. If the user selects the whole blood sample module and the probe does not detect the magnetic adapter 102, the run will stop with a warning notifying the user of an error condition.

Automated optimal shaking

The software for the flow cytometer automatically adopts a specific whole blood sample module (mode of operation) with a specific sample acquisition template. The software then sets a specific shaking speed to float only the WBCs and not the RBCs that remain bound to the bottom or sidewalls of the well.

For the agitation speed range, the agitation speed on the BioShake 3000 ranges from 100 rpm to 100 rpm to maintain the intact RBC layer drawn down by the magnetic beads while keeping the WBCs homogeneously distributed in the upper clear liquid in the sample well. 1500 rpm. Above 1500 rpm, the magnetic beads and attached RBCs start to lift into the upper clear liquid containing the WBCs. This maximum rate can be determined experimentally, for example, by observation of the top liquid color as the rate increases and when it changes from yellow to reddish. For example, once above 1500 rpm, the top liquid color starts to change from yellow to reddish or completely red.

The agitation speed range is also related to the magnetic or paramagnetic bead material and magnetic field strength disposed in the biological fluid sample. The reagent with magnetic beads used for proof-of-concept in the present invention was EasySep RBC depletion reagent (StemCell Tech, Cat. # 18170), which is a nano-magnetic bead with an estimated size between 20-500 nm (diameter) ( iron oxide, Fe ₃ O ₄ ). The magnetic beads were conjugated with an anti-human CD235 antibody to bind to the CD235 molecule expressed only on the cell surface of human RBCs. Once a magnetic field is present below the well (due to the magnets in the adapter plate), the RBC bound with the magnetic beads are pulled down to the bottom of the well.

In general, the optimal agitation speed may also be determined by the amplitude or eccentricity of the agitator itself, and therefore the optimal rotational speed of the agitator may also depend on these factors.

Automated Acquisition

The flow cytometer's software, operating in whole blood sample mode, will guide the sampling probe to descend so that the tip of the probe enters the upper liquid layer of the well, and the WBC is substantially within it without contacting or disturbing the RBC at the bottom of the well. is uniformly or homogeneously suspended. See FIGS. 6 and 1E . Sampling time and speed are limited to a certain range to avoid RBC contamination into the probe. Sampling occurs either immediately after shaking stops or during shaking, with sampling times ranging from 0.5 seconds to 5 minutes per well.

automatic analysis

After the sample probes 200 sample the wells of the microwell plate, the samples are introduced into the flow cytometer device itself, which is part of the device 400 of FIG. 9 . After passing the WBC sample through the flow cytometer instrument, the analysis software for the instrument will perform automatic data analysis. In one possible configuration, this assay includes automated bead-based sample well identification, and immune cell count normalization. The details of data analysis are not particularly relevant to the present disclosure, and algorithms known in the art and described in the patent and technical literature can be used.

Additional details of the flow cytometer and sampling device of FIGS. 9-11 can be found in U.S. Patents 10,048,191; 9,897,531; 9,797,917; and 6,890,487, the contents of which are incorporated herein by reference.

Additional optional features

The whole blood sample module software ensures that the magnetic adapter 102 is installed correctly on the shaker 104 and, when verified, locks down the shake and sampling protocols to the appropriate values. Additional cleaning can be automated in software sampling protocols. The module may also enable specific and automated assays if markers or counting beads are present. Additionally, the module may also detect RBC contamination in the sample indicating potential problems with the assay.

In one configuration, providing a permeable seal or membrane covering the wells of a microwell plate (eg, an Excel Scientific X-Pierce plate seal) that increases biosafety by preventing contamination and spillover in the event of an accident. it is possible This is particularly important for blood samples that may potentially carry unknown pathogen(s). The seal is applied to the microwell plate after reagents such as dye and magnetic beads are added to the sample and before the plate is placed on the shaker and magnetic adapter.

In another configuration, it is possible to provide in-well marker beads. These beads can be used for sample well-ID for samples with few WBCs due to certain conditions. It is also possible to provide in-well counting beads. These beads allow accurate calculation of WBC concentration based on counts of in-well counting beads. These beads may be added off-line on the table top in preliminary step A; Alternatively, they may be part of the reagents present in the rinse station 406 of FIG. 10 and introduced immediately prior to the agitation operation (Step D, FIG. 1D).

Specific assay microwell plates are contemplated for use with whole blood samples, and especially with smaller volume samples typical for 96 well plates. For example, a plate from Greiner, Cat# 675101 in a 96-well format with a well area of about half the normal well area can be used. This well geometry increases the top layer height with the same volume of sample for easy probe access, minimizes sample/reagent use, and reduces the risk of RBC contamination in the sample.

alternative embodiments

1. As described with respect to Figure 1, the primary method for isolating immune cells is negative selection of RBCs by magnetic pulling of magnetic bead-bound RBCs to the bottom of the well, where the magnet is It is placed proximal to the bottom of the well of an assay plate. An alternative location for a magnet is a gap between adjacent wells so that the magnetic field can pull the magnetic bead-bound cells to the sidewall of the assay well ( 19 in FIG. 1C ). In this configuration, the form factor and design of the magnetic adapter 102 is such that the magnetic adapter includes a feature that protrudes into a space formed in the bottom of the microwell plate 100 so that the magnets contained therein are located in the well instead of the bottom of the well. close to the side wall.

2. A second alternative method of cell isolation is the use of antibodies or affimers of interest, such as antibodies or affimers, which can bind to and float to any particular cell population on the upper surface of a liquid sample in an assay well. The use of hollow or buoyant particles conjugated to the molecule. For example, it may be possible to float RBCs to the top of a well and sample from the bottom or middle region of the well to draw WBCs into the probe. According to this design, a method for sampling immune cells in a liquid sample containing a mixture of red blood cells and immune cells includes (A) introducing hollow or buoyant particles designed to bind RBCs into the sample, (B) steps (A) before or after introducing the liquid sample into the assay wells of the microwell plate, (C) allowing the RBCs to float to the upper surface of the liquid sample in the assay wells by binding to hollow or buoyant particles, the upper surface being the WBCs and (D) taking WBCs with sampling probes from the lower or middle regions of the assay wells.

3. A third alternative method of separating cells from microwell plates uses gradient centrifugation to form multiple liquid layers in the sample in microwells, each layer containing a different cell population.

4. For all of the above different cell separation methods, after cell separation, the sampling probe descends to a specific layer containing the cell/particle population(s) of interest to acquire a sample. The sampling probe may sample only one layer by descending to a specific position in the well or may sample different layers by descending to multiple positions in the same well.

5. The sample probe may be just one single probe that descends to more than one location to acquire more than one layer. An alternative method is to use multiple probes that descend to one or more locations to acquire samples from one or more layers.

6. The method of the present disclosure can be applied to any detection system, for example, a hematology analyzer, a cell sorter, a mass spectrometer, a DNA/RNA analyzer, and the like. Thus, the method of FIGS. 1A-1E is not limited to flow cytometry. Accordingly, the device shown in FIGS. 9-11 is provided as an example and not as a limitation.

Additional Considerations

One of the applications of the method of the present invention is miniaturized "clinical-trial-in-a-dish" applications, in whole blood flow cytometry for immunology, immuno-oncology, immunotoxicity, drug profiling studies and similar research purposes. Extends the ability to directly acquire/analyze samples in a miniaturized format. The methods of the present disclosure may be used to detect RBCs or other uninteresting particles such as fluid biopsy, cerebrospinal fluid (CSF), chorionic villus sample (CVS), amniotic fluid (AF), cyst fluid sample. , can be applied to any sampling of one or more particles of interest from a mixed sample in a liquid containing a bone marrow sample, etc. For simultaneous measurement of cytokines, growth factors, chemokines, hormones, and other biological factors or particles, the mixed sample can be pre-stained with antibodies and other dyes and pre-mixed with different functional beads, such as Sartorius/IntelHcyt QBeads.

Claims (34)

A method for automatically sampling immune cells from a biological fluid sample deposited in a well of a microwell plate, wherein the well has a wall, and the biological fluid sample comprises: (1) red blood cells (RBCs); : red blood cells) and (2) magnetic beads designed to bind to the RBCs in the sample,
a) placing the microwell plate on a shaker having a magnetic adapter including at least one magnet, wherein the magnet holds the RBCs bound to the magnetic beads attached to the wall of the well , the disposing step;
b) using the shaker to spread the microwell plate substantially evenly within the biological fluid sample within the area of the well such that the immune cells are substantially isolated from the RBCs held against the wall of the well. Shaking for a certain period of time in a way to suspend the suspension (shaking); and
c) lowering a sample probe in a region of the well into the well and withdrawing a portion of the sample containing the immune cells from the region. The method of claim 1, wherein the magnetic adapter comprises a structure holding one individual magnet for each well of the microwell plate, the magnetic adapter is inserted into the upper surface of the shaker, and the upper surface and the microwell and configured to be sandwiched between plates, wherein the magnets allow the RBCs to be retained on a bottom wall or sidewall of the well. 3. The method of claim 1 or 2, wherein the biological fluid comprises whole blood. 3. The method of claim 1 or 2, wherein the biological fluid comprises cyst fluid, amniotic fluid, bone marrow sample, cerebrospinal fluid, fluid biopsy or chorionic villus sample. 5. The method according to any one of claims 1 to 4, wherein the sample probe is part of a flow cytometer and the method comprises step d) introducing a part of the sample containing the immune cells into the flow cytometer A method comprising the steps of: 6. The method of any one of claims 1 to 5, wherein the shaker further comprises a home position sensor, and the method comprises shielding the home position sensor from a magnetic field generated by the magnet. More inclusive, how. 7. The method according to any one of claims 1 to 6, wherein the shaking comprises operating the shaker in an eccentric rotation at a speed of 100 rpm to 1500 rpm. The method according to any one of claims 1 to 7, wherein the magnetic beads include magnetic or paramagnetic beads with an estimated size of 1 nm to 50 um, and bind to molecules expressed on the RBC but not to the immune cells A method that is conjugated with a specific molecule that does not bind. 9. The method according to any one of claims 1 to 8, wherein in steps a) and c) the shaker is in a shaking condition. 10. The method according to any one of claims 1 to 9, wherein the magnetic adapter comprising the at least one magnet is integrated into the shaker. 11. The method of any one of claims 1 to 10, further comprising placing a pierceable seal over the microwell plate prior to performing step a). 12. The method according to any one of claims 1 to 11, wherein the sample further comprises an in-well marker bead or an in-well counting bead. In the shaker system,
a shaker having an upper surface configured to shake a plate containing a plurality of wells in a controlled and programmable manner; and
A magnetic adapter cooperating with a structure on the shaker to removably fit onto the top surface of the shaker, the magnetic adapter comprising a structure holding an array of one or more magnets, the magnetic adapter comprising: and the magnetic adapter configured to fit on the top surface of the shaker and to be sandwiched between the top surface of the shaker and the plate. 14. The system of claim 13, wherein the magnets are arranged on the magnetic adapter to mate with a bottom portion of the well when the plate is placed on a top surface of the magnetic adapter. 14. The method of claim 13, further comprising a control system for the shaker, the control system configured to hold the plate for a period of time in such a manner that the shaker suspends the immune cells substantially equally within the biological fluid sample within the region of the well. operating the shaker to shake. 16. The system of claim 15, wherein the control system is configured to operate the agitator in eccentric rotation at a speed between 100 rpm and 1500 rpm. 17. The method of any one of claims 13 to 16, further comprising a sampling probe that is robotically controlled to move in X, Y and Z directions relative to the plate, wherein the sampling probe is immune from the well. A system in which a sample containing cells is withdrawn and the immune cells are introduced into an analysis instrument. 18. The system of claim 17, wherein the assay instrument comprises a flow cytometer. 19. The system of any one of claims 13 to 18, further comprising a ferromagnetic shield positioned between the top surface of the agitator and the magnetic adapter. 20. The system of any one of claims 13 to 19, wherein the magnetic adapter comprises an array of individual magnets, one for each well of the plate. 20. The system of any one of claims 13 to 19, wherein the magnetic adapter is configured to position the magnets in a space proximal to a sidewall of a well of the microwell plate. In the flow cytometer,
robotic sampling probe;
an agitator having an upper surface;
A magnetic adapter configured to be disposed on the top surface of the shaker, the magnetic adapter having one or more features to hold a microwell plate disposed thereon, the microwell plate comprising a plurality of wells with biological fluid samples. wherein the magnetic adapter cooperates with a structure on the shaker to be removably fitted to an upper surface of the shaker, the magnetic adapter includes a structure holding one or more magnets, the magnetic adapter comprising the shaker and the the magnetic adapter configured to be interposed between microwell plates;
A control system configured to shake the microwell plate with a shaker for a period of time in a manner to substantially evenly suspend the immune cells within the region of the wells of the microwell plate, the immune cells in the region of the wells comprising: said control system substantially insulated from red blood cells magnetically coupled to the walls of the well; and
An analytical instrumentation for performing one or more measurements on immune cells taken from the wells of the microwell plate, wherein the probes take the immune cells from the wells and introduce the immune cells into the instrument , a flow cytometer, comprising an analytical instrument. 23. The flow cytometer of claim 22, wherein the biological fluid sample comprises whole blood. 24. The flow cytometer of claims 22 or 23, wherein the biological fluid sample comprises a cyst fluid sample, amniotic fluid, bone marrow sample, cerebrospinal fluid sample, fluid biopsy or chorionic villus sample. 25. The shaker according to any one of claims 22 to 24, wherein the shaker further comprises a home position sensor, the shaker comprises a magnetic field shielding the home position sensor from a magnetic field generated by the magnet. The flow cytometer further comprising a shield positioned between the adapter and the shaker. 26. The flow cytometer of any one of claims 22 to 25, wherein the control system operates the agitator with eccentric rotation at a speed of 100 rpm to 1500 rpm. 27. The molecule of claim 26, wherein the red blood cells are bound to magnetic beads having an estimated size of 1 nm to 50 urn and are expressed on the cell surface of the red blood cells but not on the cell surface of the immune cells. A flow cytometer that is conjugated with a specific molecule that binds to. 28. The method of any one of claims 22-27, wherein the sampling probes sample a sample from the wells of the microwell plate after shaking of the microwell plate and while the immune cells are substantially uniformly suspended in the region of the well. Take out, flow cytometer. 29. The flow cytometer of claim 28, wherein the withdrawal of the sample from the well occurs at a sampling time of 0.5 seconds to 5 minutes per well. 27. The method of any one of claims 22-26, further comprising one or more reagents directed to the biological fluid sample, the one or more reagents comprising magnetic beads conjugated to antibodies designed to selectively bind to the surface of RBCs. Including, flow cytometer. A method for sampling immune cells in a liquid sample containing a mixture of red blood cells and immune cells, comprising:
(A) introducing hollow particles designed to bind to the red blood cells into the liquid sample;
(B) before or after step (A), introducing the liquid sample into an assay well of the microwell plate;
(C) floating the red blood cells in an assay to an upper surface of the liquid sample, the upper surface overlying the lower and middle regions of the assay well containing the immune cells; and
(D) extracting immune cells from the lower and/or middle region of the assay well using a sampling probe. 32. The method of claim 31, wherein the sampling probes pick up the immune cells from both the middle and bottom layers of the assay well. 32. The method of claim 31, wherein step (D) is to take immune cells from the lower region of the assay well using a first sampling probe and to take immune cells from the middle region of the assay well using a second sampling probe. , method. 34. The method of any one of claims 31-33, wherein the liquid sample comprises whole blood.

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